RU2700174C2 - Stabilized preparations containing antibodies against ngf - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and relates to a method for maintaining antibody stability in a preparation comprising storing the antibody preparation for six months or more at 5 °C or more. Antibody preparation comprises (i) about 0.1–100 mg/ml of a human antibody that specifically binds to a human nerve growth factor (hNGF); (ii) about 0.01–1.0 % polysorbate 20; (iii) about 1–20 % sucrose and (iv) about 1 mM to about 50 mM acetate.

EFFECT: invention provides a greater degree of antibody stability after storage for several months.

11 cl, 12 ex, 38 tbl

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of therapeutic antibody preparations. More specifically, the present invention relates to the field of pharmaceutical preparations comprising a human antibody that specifically binds to human nerve growth factor (NGF).

BACKGROUND OF THE INVENTION

Therapeutic macromolecules (e.g. antibodies) should be formulated as a drug in a manner that not only makes the molecules suitable for administration to patients, but also preserves their stability during storage. For example, therapeutic antibodies in a liquid solution are subject to degradation, aggregation, and / or undesirable chemical modifications, unless the solution is properly prepared. The stability of an antibody in a liquid preparation depends not only on the types of excipients used in the preparation, but also on the amounts and proportions of the excipients relative to each other. In addition, factors other than stability should be considered when preparing a liquid antibody preparation. Examples of such additional factors considered include the viscosity of the solution and the concentration of antibody that a particular drug can hold. Thus, when preparing a therapeutic antibody in the form of a drug, care must be taken to obtain a drug that remains stable, contains the appropriate concentration of the antibody and has the appropriate viscosity, as well as other properties that make it convenient to administer the drug to patients.

Antibodies against human nerve growth factor (hNGF) are one example of therapeutically relevant macromolecules that require proper preparation. Anti-NGF antibodies are clinically useful for the treatment and / or prevention of diseases such as osteoarthritis, sciatica, and other conditions such as inflammatory pain, neuropathic pain, and cancer pain.

Although anti-NGF antibodies are known, there remains a need in the art for new pharmaceutical preparations, including anti-hNGF antibodies, which are sufficiently stable and also suitable for administration to patients.

SUMMARY OF THE INVENTION

The present invention satisfies the aforementioned need by providing pharmaceutical preparations comprising a human antibody that specifically binds to human nerve growth factor (hNGF). The preparations of the present invention may include excipients in addition to antibodies against hNGF. For example, in some embodiments, the preparation may include (i) a human antibody that specifically binds to hNGF; (ii) a nonionic surfactant; and (iii) at least one carbohydrate. The nonionic surfactant may be selected from the group consisting of polysorbate 20, polysorbate 80, polyoxyethylene sorbitan monooleate and polyethylene glycol. In one embodiment, the non-ionic surfactant is Polysorbate 20. The carbohydrate may be sugar, such as, for example, sucrose, glucose, mannitol, sorbitol, lactose, or trehalose. In one embodiment, the sugar is sucrose.

In one embodiment, the pharmaceutical preparation comprises (i) about 0.1-100 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.01-1.0% polysorbate 20; and (iii) about 1-20% sucrose.

In one embodiment, the pharmaceutical preparation comprises (i) approximately 0.2-75 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.02-0.5% polysorbate 20; and (iii) about 5-10% sucrose.

In one embodiment, the pharmaceutical preparation comprises (i) about 0.6-60 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.05% polysorbate 20; and (iii) about 8% sucrose.

In some embodiments, the pharmaceutical preparation further comprises from about 1.0 mM to about 50 mM acetate.

In one embodiment, the pharmaceutical preparation comprises (i) about 1-100 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.01-1.0% polysorbate 20; and (iii) about 1-20% sucrose. In some embodiments, the pharmaceutical preparation further comprises from about 1.0 mM to about 50 mM acetate.

In one embodiment, the pharmaceutical preparation comprises (i) about 5-75 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.02-0.5% polysorbate 20; (iii) about 5-10% sucrose; and (iv) about 5-20 mM acetate.

In one embodiment, the pharmaceutical preparation comprises (i) about 6-60 mg / ml of a human antibody that specifically binds to hNGF; (ii) approximately 0.05% polysorbate 20; (iii) about 8% sucrose; and (iv) about 10 mM acetate.

In some embodiments, the preparation may also contain at least one amino acid, for example histidine or arginine. In some embodiments, the concentration of histidine or arginine can range from about 25 mM to about 100 mM.

In accordance with some variants of implementation of the present invention, the preparation is prepared in a buffer that is capable of maintaining a pH in the range from about 4.5 to about 5.6, for example, in acetate buffer.

In some embodiments, the pharmaceutical preparation exhibits a viscosity of less than about 15 centipoise, or less than about 12 centipoise, or less than about 9 centipoise.

The antibody contained in the pharmaceutical preparations of the present invention may be any antibody or fusion protein or trap that (specifically) binds specifically to hNGF. Exemplary antibodies that may be contained in the preparations of the present invention are antibodies comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising a complementarity determining heavy chain region 1 (HCDR1) having an amino acid sequence SEQ ID NO: 6, HCDR2 having the amino acid sequence of SEQ ID NO: 8, and HCDR3 having the amino acid sequence of SEQ ID NO: 10; and LCVR includes a complementarity determining region 1 light chain (LCDR1) having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 16, and LCDR3 having the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the antibody contained in the preparations of the present invention is an antibody comprising an HCVR having the amino acid sequence of SEQ ID NO: 20 and an LCVR having the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the antibody contained in the preparations of the present invention is an antibody comprising an HCVR having the amino acid sequence of SEQ ID NO: 4 and an LCVR having the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the pharmaceutical preparations may be administered intravenously or subcutaneously to a patient in need thereof. Accordingly, in some embodiments, the pharmaceutical preparation described herein can be used to prepare a medicament for treating, preventing and / or reducing the intensity of any disease or disorder associated with NGF activity or NGF activation. Exemplary, non-limiting diseases and disorders that can be treated and / or prevented by administering the pharmaceutical preparations of the present invention include pain due to any condition accompanied by neurogenic, neuropathic or nociceptive pain. In some embodiments related to neuropathic pain, the trigeminal neuralgia mentioned, postherpetic neuralgia, amputated phantom pain, fibromyalgia, sympathetic reflex dystrophy, and neurogenic pain conditions are treated. In other embodiments, cancer pain, in particular bone cancer related pain, osteoarthritis or rheumatoid arthritis pain, lumbar pain, postoperative pain at the incision site, pain at the fracture or rupture site, pain at an osteoporotic fracture, osteoporosis, joint pain with gout, diabetic nephropathy, sciatica, pain associated with sickle cell crises, migraines and other neuropathic and / or nociceptive pains are treated with the drugs described herein.

The antibody preparations of the present invention may be contained in any suitable container suitable for the storage of pharmaceutical preparations. Examples of such suitable containers include, for example, glass or plastic vials, syringes, and cartridges. The container may be transparent or opaque (for example, amber yellow). In some embodiments, the vials or syringes are coated with silicone, for example, silica. In some embodiments, the void space is filled with an inert gas, displacing any oxygen present that may adversely affect the stability of the antibody. Such an inert gas may be selected from nitrogen or argon.

In accordance with some aspects of the present invention, pharmaceutical preparations remain relatively stable after storage for several days, months, or years at a particular temperature. For example, in some exemplary embodiments of the present invention, a high percentage of the antibody (eg, 90%, 95%, 96% or more) remains in its native form after storage for at least 3, 6, 9 or more months. The percentage of the native form of the antibody can be determined, for example, using size exclusion HPLC, or using any other method known in the art. The storage temperature at which the stability of the antibody is maintained can be, for example, -80 ° C, -40 ° C, -30 ° C, -20 ° C, 0 ° C, 5 ° C, 25 ° C, 37 ° C, 45 ° C or higher.

Other embodiments of the present invention will become apparent through analysis of the following detailed description.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Before the present invention will be described, it should be understood that this invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used here is intended only to describe specific embodiments and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Except as otherwise noted, all technical and scientific terms used here have the same meaning as those usually understood by a person with average skill in the field of technology to which this invention relates. As used herein, the term “approximately”, when used with reference to a specific stated numerical value, means that the value may differ from the stated value by no more than 1%. For example, as used here, the expression "approximately 100" includes 99 and 101 and all values between them (for example, 99.1, 99.2, 99.3, 99.4, etc.).

Although in the practice or verification of the present invention, any methods and materials that are similar to or equivalent to those described herein may be used, preferred methods and materials will now be described.

PHARMACEUTICALS

As used herein, the term “pharmaceutical preparation” means a combination of at least one active ingredient (eg, a small molecule, macromolecule, compound, etc., which is capable of causing a biological effect in a human or non-human animal) and at least one an inactive ingredient which, after combining with the active ingredient and / or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. As used herein, the term “preparation” means “pharmaceutical preparation”, unless otherwise indicated. The present invention provides pharmaceutical preparations comprising at least one therapeutic polypeptide. In accordance with some embodiments of the present invention, the therapeutic polypeptide is an antibody that specifically binds to human nerve growth factor (hNGF), or an antigen binding fragment thereof. More specifically, the present invention includes pharmaceutical preparations that include (i) a human antibody that specifically binds to hNGF; (ii) a nonionic surfactant; and (iii) at least one carbohydrate. Specific, exemplary components and preparations included in the present invention are described in more detail below.

In some embodiments, the “surfactant” may be a nonionic surfactant selected from the group consisting of polysorbate 20, polysorbate 80, polyoxyethylene sorbitan monooleate, and polyethylene glycol.

In some embodiments, the pharmaceutical preparations of the present invention may be flowable preparations. As used here, the expression "fluid preparation" means a mixture of at least two components, which is advantageous in a fluid state at a temperature of from about 5 ° C to about 45 ° C. Flowable preparations include, but are not limited to, liquid preparations. Flowable preparations may be of low, medium or high viscosity, depending on their particular constituents.

ANTIBODIES THAT SPECIFICALLY CONTACT HNGF

The pharmaceutical preparations of the present invention may include a human antibody, or an antigen binding fragment thereof, which (s) specifically binds to hNGF. As used herein, the term “hNGF” means a human nerve growth factor having the amino acid sequence shown in SEQ ID NO: 2, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 1.

As used herein, the term “antibody” refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains connected via disulfide bonds, as well as their multimers (eg, IgM) ; however, the definition of the term “antibody” also includes immunoglobulin molecules consisting only of heavy chains (that is, in which there are no light chains). Each heavy chain includes a variable region of the heavy chain (here abbreviated HCVR or V _H ) and a constant region of the heavy chain. The constant region of the heavy chain includes three domains: C _H 1, C _H 2 and C _H 3. Each light chain includes a variable region of the light chain (hereinafter abbreviated LCVR or V _L ) and a constant region of the light chain. The constant region of the light chain includes one domain (CL1). The V _H and V _L regions can be further subdivided into hypervariability regions called complementarity determining regions (CDRs), distributed among regions that are more conservative, called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs located from the amino end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Except where otherwise indicated, the term “antibody”, as used herein, is intended to encompass complete antibody molecules as well as antigen-binding fragments thereof. As used herein, the terms "antigen binding portion" of an antibody, an "antigen binding fragment" of an antibody, and the like. include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen binding portion” of an antibody or “antibody fragment” refers to one or more antibody fragments that retain the ability to specifically bind to hNGF.

As used herein, the term “isolated antibody is intended to refer to an antibody essentially without other antibodies having excellent specificity for antigens (for example, an isolated antibody that specifically binds to hNGF is an antibody essentially without antibodies that specifically bind to antigens, different from hNGF).

The term “specifically binds” or the like. means that the antibody or its antigen binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may be characterized by constant dissociation of at least about 1 × 10 ^-6 M or less. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds to hNGF may have cross-reactivity with other antigens, such as NGF molecules of other species. In the context of the present invention, multispecific (eg, bispecific) antibodies that bind to hNGF, as well as to one or more additional antigens, are considered to “specifically bind” to hNGF. In addition, the isolated antibody may essentially not contain other cellular material and / or other chemicals.

Exemplary antibodies that may be included in the pharmaceutical preparations of the present invention are set forth in application US 20090041717.

In accordance with some embodiments of the present invention, the anti-hNGF antibody, or antigen binding fragment thereof, comprises a complementarity determining heavy chain region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 6, HCDR2 having the amino acid sequence of SEQ ID NO: 8, and HCDR3 having the amino acid sequence of SEQ ID NO: 10.

In accordance with some embodiments of the present invention, an anti-hNGF antibody, or antigen binding fragment thereof, comprises a complementarity determining light chain region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 16, and LCDR3 having the amino acid sequence of SEQ ID NO: 18.

In some embodiments, an anti-hNGF antibody, or antigen binding fragment thereof, comprises HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3 domains, respectively selected from the group consisting of SEQ ID NO: 6-8-10 / SEQ ID NO: 14-16-18.

In some embodiments, an anti-hNGF antibody, or antigen binding fragment thereof, comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 20. In some embodiments, an anti-hNGF antibody, or the antigen binding fragment includes a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 22. In some embodiments, an anti-hNGF antibody, or an antigen binding thereof yuschy fragment comprises HCVR / LCVR pair of amino acid sequences selected from the group consisting of SEQ ID NO: 4/12 and 20/22.

The non-limiting, exemplary antibody used herein in the examples is called “mAb1". This antibody is also referred to in US20090041717 and includes, as described here, a pair of HCVR / LCVR amino acid sequences having SEQ ID NO: 20/22 and the HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3 domains represented by SEQ ID NO: 6 -8-10 / SEQ ID NO: 14-16-18.

The amount of antibody, or antigen-binding fragment thereof, contained in the pharmaceutical preparations of the present invention may vary depending on the specific desired properties of the drugs, as well as on the specific circumstances and purposes for which the drugs are intended to be used. In some embodiments, the pharmaceutical preparations may contain from about 0.01 mg / ml to about 500 mg / ml of an antibody; from about 1 mg / ml to about 500 mg / ml of an antibody; from about 5 mg / ml to about 400 mg / ml of an antibody; from about 5 mg / ml to about 200 mg / ml of an antibody; from about 25 mg / ml to about 180 mg / ml of an antibody; from about 25 mg / ml to about 150 mg / ml of an antibody; or from about 50 mg / ml to about 180 mg / ml of an antibody. In some embodiments, the pharmaceutical preparations may contain from about 0.1 mg / ml to about 100 mg / ml of an antibody, from about 0.2 mg / ml to about 75 mg / ml of an antibody, from about 0.6 mg / ml to about 60 mg / ml antibody. For example, the preparations of the present invention may contain approximately 0.1 mg / ml, approximately 0.2 mg / ml, approximately 0.6 mg / ml, approximately 1 mg / ml, approximately 2 mg / ml, approximately 5 mg / ml, approximately 10 mg / ml, approximately 15 mg / ml, approximately 20 mg / ml, approximately 25 mg / ml, approximately 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml, approximately 75 mg / ml, approximately 80 mg / ml, approximately 85 mg / ml, approximately 86 mg / ml, approximately 87 mg / ml, approximately 88 mg / ml, approximately 89 mg / ml, approximately 90 mg / ml, approximately 95 mg / ml, approximately 100 mg / ml, approximately 105 mg / ml, approximately 110 mg / ml, approximately 115 mg / ml, approximately 120 mg / ml, approximately 125 mg / ml, approximately 130 mg / ml, approximately 131 mg / ml, approximately 132 mg / ml approximately 133 mg / ml, approximately 134 mg / ml, approximately 135 mg / ml, approximately 140 mg / ml, approximately 145 mg / ml, approximately 150 mg / ml, approximate about 155 mg / ml, about 160 mg / ml, about 165 mg / ml; approximately 170 mg / ml; approximately 175 mg / ml; approximately 180 mg / ml; approximately 185 mg / ml; approximately 190 mg / ml, approximately 195 mg / ml, or approximately 200 mg / ml of the antibody or antigen binding fragment thereof that specifically binds to hNGF.

FILLERS and pH

The pharmaceutical preparations of the present invention include one or more excipients. As used herein, the term “excipient” means any non-therapeutic agent added to the preparation to provide the desired consistency, viscosity or stabilization effect.

In some embodiments, the pharmaceutical preparation of the present invention comprises a buffer suitable for maintaining a pH in the range of about 4.5 to about 5.6. An exemplary buffer suitable for use in the preparations of the present invention includes, for example, acetate buffer. In one embodiment, the acetate buffer is prepared at a concentration of 10 mM.

The amount of acetate contained in the pharmaceutical preparations of the present invention can vary from about 1 mM to about 50 mM, from about 2 mM to about 20 mM, from about 3 mM to about 12 mM, or about 10 mM.

The pharmaceutical preparations of the present invention may also include one or more carbohydrates, for example, one or more sugars. Sugar can be reducing sugar or non-reducing sugar. “Reducing sugars” include, for example, sugars with a ketone or aldehyde group and contain a reactive semi-acetal group that allows the sugar to function as a reducing agent. Specific examples of reducing sugars include fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, ramnose, galactose and maltose. Non-reducing sugars may include anomeric carbon, which is acetal and essentially does not react with amino acids or polypeptides to initiate the Maillard reaction. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, sorbitol, melecitose, and raffinose. Sugar acids include, for example, sugar acids, gluconate and other multi-hydroxy sugars and their salts.

The amount of sugar contained in the pharmaceutical preparations of the present invention may vary depending on the specific circumstances and intended purposes for which the preparations are used. In some embodiments, the preparations may contain from about 0.1% to about 20% sugar, from about 0.5% to about 20% sugar, from about 1% to about 20% sugar, from about 2% to about 15% sugar from about 3% to about 10% sugar, from about 4% to about 10% sugar, or from about 5% to about 10% sugar. For example, the pharmaceutical preparations of the present invention may contain approximately 0.5%, approximately 1.0%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 3.5%, approximately 4.0%, approximately 4.5%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8 0%, approximately 8.5%, approximately 9.0%, approximately 9.5%, approximately 10.0%, approximately 10.5%, approximately 11.0%, approximately 11.5%, approximately 12.0 %, approx 12.5%, approximately 13.0%, approximately 13.5%, approximately 14.0%, approximately 14.5%, approximately 15.0%, approximately 15.5%, approximately 16.0%, 16, 5%, approximately 17.0%, approximately 17.5%, approximately 18.0%, approximately 18.5%, approximately 19.0%, approximately 19.5%, or approximately 20.0% sugar (e.g. sucrose) .

The pharmaceutical preparations of the present invention may also include one or more surfactants. As used herein, the term “surfactant” means a substance that reduces the surface tension of a liquid in which it is dissolved and / or reduces the interfacial tension between oil and water. Surfactants may be ionic or non-ionic. Exemplary non-ionic surfactants that may be included in the preparations of the present invention include, for example, alkyl polyethylene oxide, alkyl polyglucosides (e.g. octyl glucoside and decylmaltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, MEA cocamide, cocamide DEA and cocamide tea. Specific nonionic surfactants that may be included in the preparations of the present invention include, for example, polysorbates such as Polysorbate 20, Polysorbate 28, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80, Polysorbate 81 and Polysorbate 85; poloxamers, such as poloxamer 188, poloxamer 407; polyethylene-polypropylene glycol or polyethylene glycol (PEG). Polysobate 20 is also known as TWEEN 20, sorbitol monolaurate and polyoxyethylene sorbitan monolaurate.

The amount of surfactant contained in the pharmaceutical preparations of the present invention may vary depending on the specific desired properties of the preparations, as well as on the specific circumstances and purposes for which the preparations are intended to be used. In some embodiments, the preparations may contain from about 0.01% to about 10% surfactant, from about 0.05% to about 5% surfactant, or from about 0.1% to about 1% surfactant substances. For example, the preparations of the present invention may contain approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.10%, approximately 0.11%, approximately 0.12%, approximately 0.13%, approximately 0.14%, approximately 0.15%, approximately 0, 16%, approximately 0.17%, approximately 0.18%, approximately 0.19%; approximately 0.20%, approximately 0.21%, approximately 0.22%, approximately 0.23%, approximately 0.24%, approximately 0.25%, approximately 0.26%, approximately 0.27%, approximately 0 , 28%, approximately 0.29%, or approximately 0.30% of a surfactant (e.g., Polysorbate 20).

The pharmaceutical preparations of the present invention may have a pH in the range of from about 4.0 to about 6.0. For example, the preparations of the present invention can have a pH of approximately 4.2, approximately 4.4, approximately 4.6, approximately 4.8, approximately 5.0, approximately 5.2, approximately 5.4, approximately 5.6, about 5.8 or about 6.0.

PRESENTED AS AN EXAMPLE OF DRUGS

In accordance with one aspect of the present invention, the pharmaceutical preparation comprises (i) a human antibody that specifically binds to hNGF (eg, mAb1); (ii) acetate; and (iii) sugar (e.g., sucrose). Specific, non-limiting, exemplary embodiments encompassed by this aspect of the present invention are set forth in Table 1.

Table 1 Exemplary Pharmaceutical Preparations Including mAb1, Acetate, and Sucrose mAb1 (mg / ml) 5 5 10 fifteen 5 5 10 fifteen 5 5 10 fifteen 5 5 10 fifteen 0 0 0 0 0 0 0 0 0 0 0 0 acetate (mm) one
0 one
0 10 10 one
0 one
0 10 10 one
0 one
0 10 10 one
0 one
0 10 10 sucrose (%) 2 2 2 2 four four four four 6 6 6 6 8 8 8 8

In accordance with another aspect of the present invention, a pharmaceutical preparation includes (i) a human antibody that specifically binds to hNGF (e.g., mAb1), (ii) acetate, (iii) sugar (e.g., sucrose), and (iv) a surfactant ( e.g. polysorbate 20). Specific, non-limiting, exemplary embodiments covered by this aspect of the present invention are set forth in Tables 2A and 2B.

Table 2A Exemplary pharmaceutical preparations including mAb1, acetate, sucrose and polysorbate 20 mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Table 2B Exemplary pharmaceutical preparations including mAb1, acetate, sucrose and polysorbate 20 mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

In accordance with another aspect of the present invention, a pharmaceutical preparation includes (i) a human antibody that specifically binds to hNGF (e.g., mAb1), (ii) acetate, (iii) sugar (e.g., sucrose); (iv) a surfactant (e.g., polysorbate 20), (v) a first amino acid (e.g., histidine), and (v) a second amino acid (e.g., arginine). Specific, non-limiting, exemplary embodiments covered by this aspect of the present invention are set forth in Tables 3A, 3B, 3C, 3D, 3E, and 3F.

Table 3A Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 histidine (mm) 10 10 10 10 10 10 10 10 10 10 10 10 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 arginine (mm) 10 10 10 10 10 10 10 10 10 10 10 10

Table 3B Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 histidine (mm) 10 10 10 10 10 10 10 10 10 10 10 10 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 arginine (mm) 25 25 25 25 25 25 25 25 25 25 25 25

Table 3C Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 histidine (mm) 25 25 25 25 25 25 25 25 25 25 25 25 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 arginine (mm) 10 10 10 10 10 10 10 10 10 10 10 10

3D table Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 histidine (mm) 25 25 25 25 25 25 25 25 25 25 25 25 sucrose (%) 2 2 2 2 four four four four 8 8 8 8 polysorbate 20 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 arginine (mm) 25 25 25 25 25 25 25 25 25 25 25 25

Table 3E Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 Acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 Histidine (mm) 25 25 25 25 25 25 25 25 25 25 25 25 Sucrose (%) 2 2 2 2 5 5 5 5 10 10 10 10 polysorbate 20 (%) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 arginine (mm) fifty fifty fifty fifty fifty fifty fifty fifty fifty fifty fifty fifty

Table 3F Exemplary pharmaceutical preparations including mAb1, acetate, histidine, sucrose, polysorbate 20 and arginine mAb1 (mg / ml) 5 fifty one hundred 150 5 fifty one hundred 150 5 fifty one hundred 150 Acetate (mm) 10 10 10 10 10 10 10 10 10 10 10 10 Histidine (mm) 25 25 25 25 25 25 25 25 25 25 25 25 Sucrose (%) 2 2 2 2 5 5 5 5 10 10 10 10 polysorbate 20 (%) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 arginine (mm) 25 25 25 25 25 25 25 25 25 25 25 25

In accordance with some aspects of the present invention, an antibody preparation may include an anti-hNGF antibody at a concentration of about 20 mg / ml in about 10 mM acetate buffer, plus about 1.0% polyethylene glycol 3350 (PEG 3350) and about 20% sucrose at pH equal to approximately 5.0. In one embodiment, the antibody preparation is administered intravenously. In one embodiment, the antibody preparation is administered subcutaneously.

Additional, non-limiting examples of pharmaceutical preparations covered by the present invention are set forth elsewhere herein, including the demo examples below.

STABILITY AND VISCOSITY OF PHARMACEUTICALS

The pharmaceutical preparations of the present invention typically exhibit high levels of stability. The term “stable” as used herein with respect to pharmaceutical preparations means that a satisfactory degree of structure and / or function and / or biological activity remains in the antibodies in pharmaceutical preparations after storage for a predetermined period of time. The drug can be stable even if the antibody contained in it does not retain 100% of its structure and / or function and / or biological activity after storage for a predetermined period of time. In some cases, the retention of approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% of the structure and / or function and / or biological activity of the antibody after storage for a given period of time can be considered as a "stable drug."

Stability can be measured, inter alia, by determining the percentage of the native form of the antibody remaining in the preparation after storage for a predetermined period of time at a particular temperature. The percentage of the native form of the antibody can be determined using, among other things, size exclusion chromatography (eg, high performance size exclusion chromatography [exclusion HPLC]). A “satisfactory degree of stability,” as this expression is used here, means that at least 90% of the native form of the antibody can be detected in the preparation after storage for a predetermined period of time at a particular temperature. In some embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native form of the antibody can be detected in the preparation after storage in the course of a given period of time at a specific temperature. The predetermined period of time after which stability is determined may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. The temperature at which a pharmaceutical preparation can be stored in a stability assessment can be any temperature in the range of about -80 ° C to about 45 ° C, for example, storage at about -30 ° C, about -20 ° C, about 0 ° C , approximately 5 ° C, approximately 25 ° C, approximately 37 ° C, or approximately 45 ° C. For example, a pharmaceutical preparation can be considered stable if, after storage for 3 months at 5 ° C, more than approximately 90%, 95%, 96%, or 97% of the native form of the antibody is detected by size exclusion HPLC. A pharmaceutical preparation can also be considered stable if, after storage for 6 months at 5 ° C, more than approximately 90%, 95%, 96% or 97% of the native form of the antibody is detected by size exclusion HPLC. A pharmaceutical preparation can also be considered stable if, after storage for 9 months at 5 ° C, more than approximately 90%, 95%, 96% or 97% of the native form of the antibody is detected by exclusion HPLC. A pharmaceutical preparation can also be considered stable if, after storage for 3 months at 25 ° C, more than approximately 90%, 95%, 96% or 97% of the native form of the antibody is detected by exclusion HPLC. A pharmaceutical preparation can also be considered stable if, after storage for 6 months at 25 ° C, more than approximately 90%, 95%, 96% or 97% of the native form of the antibody is detected by exclusion HPLC. A pharmaceutical preparation can also be considered stable if, after storage for 9 months at 25 ° C, more than approximately 90%, 95%, 96% or 97% of the native form of the antibody is detected by exclusion HPLC.

Other methods can be used to evaluate the stability of the preparations of the present invention, such as differential scanning calorimetry (DSC) for determining thermal stability, adjustable mixing for determining mechanical stability, and determining optical density at approximately 350 nm or approximately 405 nm for determining turbidity of solutions. For example, the preparation of the present invention can be considered stable if, after storage for 6 or more months at about 5 ° C to about 25 ° C, the change in OD _{405 of the} drug is less than about 0.05 (for example, 0.04, 0.03, 0.02, 0.01 or less) compared with OD _{405 of the} drug at t = 0.

Stability can also be assessed by measuring the biological activity and / or affinity of binding of the antibody to its target. For example, the preparation of the present invention can be considered stable if, after storage at, for example, 5 ° C, 25 ° C, 37 ° C, 45 ° C, etc. over a given period of time (e.g. 1-12 or 18 months), the anti-hNGF antibody contained in the preparation binds to hNGF with an affinity of at least 50%, 60%, 70%, 80%, 90%, 95 % or more of the binding affinity of the antibody to the indicated storage. Additional methods for assessing the stability of antibodies in a preparation are demonstrated in the Examples section below.

In some embodiments, the pharmaceutical preparations of the present invention in fluid form exhibit low to medium viscosity levels. The “viscosity” as used here may be “kinematic viscosity” or “absolute viscosity”. “Kinematic viscosity” is an indicator of the resistance of a fluid to flow under the influence of gravity. When two liquids of equal volume are placed in identical capillary viscometers and allowed to flow under the action of gravity, the flow of a viscous liquid through a capillary is longer than that of a less viscous liquid. For example, if the full flow of one fluid takes 200 seconds, and that of another fluid takes 400 seconds, the second fluid is twice as viscous as the first fluid on a kinematic viscosity scale. “Absolute viscosity”, sometimes called dynamic viscosity or simply viscosity, is the product of kinematic viscosity and fluid density (Absolute viscosity = kinematic viscosity × density). Kinematic viscosity is measured in L ² / T, where L is the length and T is the time. Typically, kinematic viscosity is presented in centistokes (cSt). The unit of kinematic viscosity in the SI system is mm ² / sec, which is equal to 1 cSt. Absolute viscosity is presented in units of centipoise (cP). The absolute viscosity unit in the SI system is milliPascal second (MPa-sec), where 1 cP = 1 MPa-sec.

As used herein with respect to the fluid preparation of the present invention, an absolute viscosity of less than about 20 centipoise (cP) will be demonstrated at a low viscosity level. For example, it is believed that the flowable preparation of the present invention has a “low viscosity” if, after measurement using standard viscosity measurement methods, the preparation exhibits an absolute viscosity of approximately 19 cP, approximately 18 cP, approximately 17 cP, approximately 16 cP, approximately 15 cP, approximately 14 cP, approximately 13 cP, approximately 12 cP, approximately 11 cP, approximately 10 cP, approximately 9 cP, approximately 8 cP, approximately 7 cP, approximately 6 cP, approximately 5 cP, approximately Tel'nykh 4 cps or less. As used herein with respect to the fluid preparation of the present invention, with an average viscosity level, an absolute viscosity of between about 30 cP and about 20 cP will be demonstrated. For example, it is believed that the flowable preparation of the present invention has an “average viscosity” if, after measurement using standard viscosity measurement methods, the preparation exhibits an absolute viscosity of approximately 30 cP, approximately 29 cP, approximately 28 cP, approximately 27 cP, approximately 26 cP, approximately 25 cP, approximately 24 cP, approximately 23 cP, approximately 22 cP, approximately 21 cP, or approximately 20 cP.

As demonstrated in Example 5 below, the inventors of the present invention unexpectedly found that low to medium viscosity flowable preparations containing high concentrations of anti-hNGF antibodies (for example, up to at least 125-150 mg / ml) can be obtained by preparing the antibody using 25 mM histidine and 25 mM-100 mM arginine. In addition, it was further discovered that the viscosity of the preparation can be reduced even more by lowering the sucrose content.

CONTAINERS FOR PHARMACEUTICALS AND ADMINISTRATION METHODS

The pharmaceutical preparations of the present invention may be contained in any container suitable for storing drugs and other therapeutic compositions. For example, pharmaceutical preparations may be contained in a sealed and sterilized plastic or glass container of a predetermined volume, such as a vial, ampoule, syringe, cartridge or bottle. Various types of vials can be used to hold the preparations of the present invention, including, for example, transparent and opaque (eg, amber-yellow) glass or plastic bottles. Also, any type of syringe can be used to hold and / or administer the pharmaceutical preparations of the present invention. The pharmaceutical preparations in the container can be processed using any method known in the art for removing oxygen to increase the stability of the antibody, if necessary. Oxygen in the free space (the gas space above the liquid in a closed container) can be replaced with an inert gas such as nitrogen or argon.

The pharmaceutical preparations of the present invention may be contained in syringes with a conventional tungsten content or in syringes with a low tungsten content. As will be appreciated by those of ordinary skill in the art, the manufacturing process for glass syringes typically involves the use of a hot tungsten rod, the function of which is to push a hole in the glass, thereby creating a hole from which liquids can be removed and expelled from the syringe. This process leads to the deposition of small amounts of tungsten on the inner surface of the syringe. To reduce the amount of tungsten in the syringe, subsequent washing steps and other treatments can be used. The term “conventional tungsten” as used herein means that the syringe contains more than 500 parts per billion (ppm) of tungsten. The term “low tungsten” means that the syringe contains less than 500 ppm of tungsten. For example, a low tungsten syringe according to the present invention may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100 , 90, 80, 70, 60, 50, 40, 30, 20, 10 or less ppm of tungsten.

The rubber sealing cuffs on the piston used in the syringes and the rubber stoppers used to close the openings of the vials may be coated with a layer to prevent contamination of the medicinal contents of the syringe or vial and / or to maintain its stability. Thus, in accordance with some embodiments, the pharmaceutical preparations of the present invention may be contained in a syringe that includes a coated cuff on a piston, or in a vial that is sealed with a coated rubber stopper. For example, a cuff or cork may be coated with a thin layer of fluorocarbon. Examples of coated plugs and / or cuffs suitable for use with vials and syringes containing pharmaceutical preparations of the present invention are mentioned, for example, in US Pat. Nos. 4,997423, 5908686, 6286699, 6645635 and 7226554. Specific, exemplary coated a layer of rubber stoppers and cuffs that may be used with the present invention are commercially available under the trademark FluroTec® from West Pharmaceutical Services, Inc. (Lionville, PA).

According to some embodiments of the present invention, the pharmaceutical preparations may be contained in a low tungsten syringe that contains a cuff coated with a layer of fluorocarbon. However, as demonstrated in the Examples section below, the anti-NGF antibody of the present invention appears to be stable in any of the tested syringe and cuff combinations.

Pharmaceutical preparations can be administered to a patient parenterally, for example, by injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or by transdermal administration, mucosal administration, intranasal, pulmonary and / or oral administration. Numerous pens and / or reusable auto-delivery devices may be used for subcutaneous delivery of the pharmaceutical preparations of the present invention. Examples include, but are not limited to, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, United Kingdom), DISETRONIC ™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25 ™ pen, HUMALOG ™ pen, HUMALIN 70 pen / 30 ™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN ™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), BD ™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN ™, OPTIPEN PRO ™, OPTIPEN STARLET ™ and OPTICLIK ™ (Sanofi-Aventis, Frankfurt, Germany), not to mention others . Examples of disposable delivery devices and / or auto-injectors for subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR ™ (Sanofi-Aventis), FLEXPEN ™ (Novo Nordisk) and KWIKPEN ™ (Eli Lilly), SURECLICK ™ Auto Injector (Amgen, Thousand Oaks, CA), PUSHCLICK ™ (Scandinavian Health, Ltd. (SHL) Group), PENLET ™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and the HUMIRA ™ pen (Abbott Labs, Abbott Park, IL), not to mention others.

Also provided is the use of a micro-infusion device for delivering pharmaceutical preparations of the present invention. As used herein, the term “microinfusion device” means a subcutaneous delivery device designed to slowly administer large volumes (eg, up to about 2.5 ml or more) of a therapeutic drug over an extended period of time (eg, about 10, 15, 20, 25 , 30 minutes or more). See, for example, US Pat. Nos. 6629949 and 6659982 and Meehan et al., J. Controlled Release 46: 107-116 (1996). Microinfusion devices are particularly useful for delivering large doses of therapeutic proteins contained in high concentrations (e.g., approximately 100, 125, 150, 175, 200 or more mg / ml) and / or viscous solutions.

THERAPEUTIC APPLICATIONS OF PHARMACEUTICAL DRUGS

The pharmaceutical preparations of the present invention are useful, inter alia, for treating, preventing and / or reducing the intensity of any disease or disorder associated with NGF activity. Exemplary, non-limiting diseases and disorders that can be treated and / or prevented by administering the pharmaceutical preparations of the present invention include pain due to any condition accompanied by neurogenic, neuropathic or nociceptive pain. In some embodiments related to neuropathic pain, the trigeminal neuralgia mentioned, postherpetic neuralgia, amputated phantom pain, fibromyalgia, sympathetic reflex dystrophy, and neurogenic pain conditions are treated. In other embodiments, cancer pain, in particular bone cancer related pain, osteoarthritis or rheumatoid arthritis pain, lumbar pain, postoperative pain at the incision site, pain at the site of fracture or rupture, pain during osteoporotic fracture, osteoporosis, joint pain with gout, diabetic nephropathy, sciatica, pain associated with sickle cell crises, migraines and other neuropathic and / or nociceptive pains are preferably treated. Thus, the present invention includes methods of treating, preventing and / or reducing the intensity of any disease or disorder associated with NGF activity or activation of NGF (including any of the above, cited as examples of diseases, disorders and conditions) through the use of pharmaceutical preparations of the present inventions. The therapeutic methods of the present invention include administering to a subject any preparation comprising an anti-hNGF antibody disclosed herein. The subject to whom the pharmaceutical preparation is administered may be, for example, any person or non-human animal that needs such treatment, prevention and / or reduction of intensity, or one who would otherwise benefit from the inhibition or attenuation of NGF activity and / or NGF-mediated activity. For example, the subject may be an individual who is diagnosed with any of the above diseases or disorders, or who is considered to be at risk of being affected. The present invention further includes the use of any of the pharmaceutical preparations disclosed herein for the manufacture of a medicament for treating, preventing and / or reducing the intensity of any disease or disorder associated with NGF activity or NGF activation (including any of the foregoing as an example of diseases, disorders and conditions).

EXAMPLES

The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and apply the methods and compositions of the present invention, and are not intended to limit the scope of what the present inventors consider as an invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), but some experimental errors and deviations should be considered. Except where otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is presented in degrees Celsius, and pressure is near or near atmospheric.

Example 1. The stability of a fully human antibody against human nerve growth factor (NGF) ("mAb1") after storage at low temperatures

In this example, various formulations were prepared containing an anti-human NGF antibody without bulking agents. An exemplary antibody used in this and in all subsequent examples set forth below is an antibody comprising a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 20 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: 22. This antibody is referred to herein as “mAb1".

As in the preliminary experiment, the stability of mAb1 in a liquid solution was determined after various periods of time of storage in a frozen state at -30 ° C and -80 ° C. The concentration of mAb1 used in this example was 130 mg / ml. At various points in time, the stability of mAb1 was determined using high-resolution liquid chromatography (HPLC) or high-resolution cation-exchange liquid chromatography (cation-exchange HPLC). Stability was estimated based on the percentage of the native form of mAb1 remaining in the sample (using size exclusion HPLC; table 4) and the percentage of acid variant noted in the sample (using cation exchange HPLC; table 5). An increase in the percentage of the acid variant is consistent with deamidation of the antibody and is therefore considered undesirable with respect to the pharmaceutical preparations of the present invention.

Table 4 Remaining% native form mAb1 (exclusion HPLC) Storage temperature Time (months) -80 ° C -30 ° C 0 96.6 96.6 one 96.6 95.2 3 96.6 94.6 6 96.6 94.2 9 96.5 93.1 12 96.7 93.3

Table 5 % acid variant (cation exchange HPLC) Storage temperature Time (months) -80 ° C -30 ° C 0 14.3 14.3 one 14.7 14.6 3 14.9 14.5 6 14.1 12.8 9 14.9 14.5 12 14.6 14.6

These results show that mAb1 can remain stable at a concentration of 130 mg / ml for at least 12 months when stored at -80 ° C.

Example 2. The stability of the preparations of mAb1 containing a minimum amount of fillers.

Six different preparations containing mAb1 were prepared at a concentration of 40 mg / ml with a minimum amount of excipients (as shown in table 6, see also example 2) and stored at -20 ° C or -30 ° C for various periods time. All preparations contain 10 mM acetate, pH 5.0. Table 7 (-30 ° C) and table 8 (-20 ° C) show the percentage of the native form of mAb remaining in various formulations with a minimum amount of excipients as determined by size exclusion HPLC.

Table 6 Mab1 preparations containing a minimum amount of fillers A drug Filler mAb1 (mg / ml) one 0.5% polyethylene glycol 3350 40 2 1.0% polyethylene glycol 3350 40 3 1% Sucrose 40 four 2% Sucrose 40 5 4% Sucrose 40 6 no 40

As noted above, drug stability was checked using size exclusion HPLC after storage for various time periods at -30 ° C and -20 ° C. The results, presented as the remaining percentage of the native form of mAb1, are shown in Table 7 (when stored at -30 ° C) and 8 (-20 ° C).

Table 7 Remaining% native form mAb1 (exclusion HPLC) after storage at -30 ° C # of the drug (see table 6) Time (months) one 2 3 four 5 6 0 98.3 98.1 98.0 97.9 98.1 98.0 one 98.0 98.1 98.0 98.0 98.0 97.0 3 97.8 97.9 98.0 98.0 97.9 96.0 6 98.2 98.4 98.5 98.4 98.4 95.9 9 98.2 98.3 98.5 98.5 98.8 95.1 12 98.2 98.4 98.4 98.4 98.5 95.7 eighteen 97.7 98.2 98.1 98.1 98.4 94.5 24 97.9 98.1 98.3 98.5 98.5 94.5

Table 8 Remaining% native form mAb1 (exclusion HPLC) after storage at -20 ° C # of the drug (see table 6) Time (months) one 2 3 four 5 6 0 98.3 98.1 98.0 97.9 98.1 98.0 one 97.8 98.1 98.2 98.1 98.2 95.8 3 97.8 97.9 98.1 98.2 98.0 93.0 6 97.7 98.3 98.3 98.4 98.5 92.6 9 97.8 98.1 98.3 98.3 98.3 90.7 12 97.7 98.2 98.3 98.3 98.5 92.9 eighteen 96.4 97.7 97.9 98.1 98.3 86.7 24 96.6 97.9 98.1 98.2 98.3 88.6

As shown in this example, the stability of mAb1 is largely maintained in preparations 1, 2, 3, 4, and 5 after storage for several months at -20 ° C and -30 ° C. These results indicate that the stability of mAb1 at -30 ° C can be increased by adding at least 1.0% sucrose or at least 0.5% polyethylene glycol 3350. These results further indicate that the stability of mAb1 at -20 ° C, it can be increased by adding at least 1.0% sucrose or at least 1.0% polyethylene glycol 3350.

Example 3. The stabilized drug mAb1

For use in Examples 4 and 5 below, a stabilized preparation was prepared containing various concentrations of mAb1. This drug, called “drug A”, is presented in table 9.

Table 9 The stabilized preparation "A" mAb1 Component Drug A mAb1 6-100 mg / ml Acetate Polysorbate 20 0.05% Sucrose 8% pH 5,0

Example 4. The stability of the drug A after storage at 5 ° C

The stability of preparation A (see Example 3) containing 6, 20 or 100 mg / ml mAb1 was tested after storage for several months at 5 ° C in clear glass vials. Stability was evaluated by the following parameters: (a) appearance; (b) turbidity (OD 405 nm); (c) pH; (d) the percentage of total mAb recovered as determined by reverse phase HPLC; (e) the percent recoverable native form of mAb1 (as determined by size exclusion HPLC); (f) the percentage of the main peak of recovered mAb1 (determined using cation exchange HPLC); and (g) the percentage of recoverable acidic variant mAb1 (determined using cation exchange HPLC). The stability assessment results of preparation A containing 6, 20 and 100 mg / ml mAb1 are summarized in Tables 10, 11 and 12, respectively.

Table 10 The stability of drug A, containing 6 mg / ml mAb1, after storage at 5 ° C in glass vials (0-12 months) Duration of storage at 5 ° C (months) Parameter 0 one 2 3 6 9 12 Appearance Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Turbidity (OD 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PH 5.1 5.1 5.1 5,0 5,0 5.1 5.1 % recovered mAb1 in general one hundred 96 101 99 102 107 107 % recoverable native form mAb1 (exclusion HPLC) 98.8 98.6 98.7 98.9 98.5 98.8 98.7 % of the main peak recovered mAb1 (cation exchange HPLC) 44.8 46.3 46,4 454 41.2 46.2 45.0 % recoverable acid variant mAb1 (cation exchange HPLC) 16.6 16.5 17.4 17.7 15,2 15.9 16.5

Table 11 The stability of drug A containing 20 mg / ml mAb1, after storage at 5 ° C in glass vials (0-18 months) Duration of storage at 5 ° C (months) Parameter 0 one 2 3 6 9 12 eighteen Appearance Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Turbidity (OD 405 nm) 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 pH 5.1 5.2 5,0 5.1 5,0 5.1 5.2 5.1 % recovered mAb1 in general one hundred 96 99 one hundred one hundred 104 101 102 % recoverable native form mAb1 (exclusion HPLC) 99.0 98.4 98.5 98.4 98.6 98.2 98.6 98.2 % of the main peak recovered mAb1 (cation exchange HPLC) 44.0 44.0 44.1 43.8 44.3 43.8 44.9 43.6 % recoverable acid variant mAb1 (cation exchange HPLC) 15.8 15,5 15.8 15,5 15,5 15,2 15.9 15.3

Table 12 The stability of drug A containing 100 mg / ml mAb1, after storage at 5 ° C in glass vials (0-18 months) Duration of storage at 5 ° C (months) Parameter 0 one 2 3 6 9 12 eighteen Appearance Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Satisfied Turbidity (OD 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 pH 5,0 5.1 5,0 5.1 5,0 5.1 5,0 5.2 % recovered mAb1 in general one hundred 98 106 104 103 103 101 104 % recoverable native form mAb1 (exclusion HPLC) 98.2 97.9 97.5 97.8 98.2 97.4 98.1 97.6 % of the main peak recovered mAb1 (cation exchange HPLC) 41.3 40.9 39.2 39.0 39.5 39.7 42.0 40,4 % recoverable acid variant mAb1 (cation exchange HPLC) 14.8 14.9 13.7 14.0 14.0 13,4 13.8 14.0

The results of this example show that preparation A containing 6 mg / ml mAb1 remained stable for at least 12 months storage at 5 ° C in clear glass vials, while approximately 98.7% or more of the native form of mAb1 remained in the samples after storage for 12 months under such conditions. The results of this example also show that formulation A containing 20 mg / ml mAb1 remained stable for at least 18 months storage at 5 ° C in clear glass vials, while approximately 98.2% of the native form of mAb1 remained in samples after storage for 18 months under such conditions. In addition, preparation A containing 100 mg / ml mAb1 remained stable for at least 18 months of storage at 5 ° C in clear glass vials, while approximately 97.6% of the native form of mAb1 remained in the samples after storage for 18 months in such conditions. In addition, the percentage of the acid variant has not changed significantly since time = 0 after storage for 12 or 18 months under the studied conditions, thereby confirming the stability of the preparations.

Example 5. The effect of concentrations of arginine, histidine and sucrose on the viscosity and stability of preparations containing mAb1.

Several preparations were prepared containing 100 mg / ml, 125 mg / ml and 150 mg / ml mAb1 and various amounts of histidine, arginine and sucrose. For each preparation, viscosity and osmolality were determined at 25 ° C. The results are summarized in table 13.

Table 13 Effect of arginine, histidine and sucrose on the viscosity of preparations containing mAb1 (All preparations contain 10 mM acetate, pH 5.0 and 0.2% polysorbate 20 mAb1 (mg / ml) Histidine (mm) Histidine (mm) Sucrose
(%) Viscosity (Centipoise) Osmolality (milliosmol) one hundred 0 0 10 -13.5 440 one hundred 0 25 10 ~ 9 510 one hundred 25 0 10 ~ 9 510 one hundred 0 25 5 ~ 7 270 one hundred 0 fifty 10 ~ 7 560 125 0 0 5 -twenty -250 125 0 25 5 ~ 11 290 125 25 0 5 -12 280 125 0 fifty 5 ~ 9 340 125 25 25 5 ~ 9 330 125 0 0 2 -16 150 150 0 0 5 ~ 41 300 150 25 25 5 -19 360 150 25 25 2 -fifteen 310 150 0 75 5 -fourteen 450 150 0 one hundred 5 -eleven 510

The results presented in table 13 indicate that the addition of histidine or arginine at a concentration of 25 mM to mAb1 (at a concentration of 100 mg / ml) significantly reduced the viscosity of the drug compared to an antibody preparation containing neither histidine nor arginine . In addition, the addition of arginine at a concentration of 25 mM to mAb1 (at a concentration of 100 mg / ml), with a decrease in sucrose concentration from 10% to 5%, led to a further decrease in viscosity. When the concentration of mAb1 antibodies was increased to 125 mg / ml, both histidine and arginine at a concentration of 25 mM resulted in a significant decrease in viscosity when used individually or together. In addition, a decrease in sucrose concentration from 5% to 2% with the addition of histidine and arginine reduced the viscosity of the drug to an even greater extent. When the concentration of mAb1 was increased to 150 mg / ml, the combination of histidine and arginine, each at a concentration of 25 mM, resulted in a significant decrease in viscosity. A decrease in sucrose from 5% to 2% led to a further decrease in viscosity.

Based, at least in part, on the foregoing, the following preparation “B” was prepared, as presented in table 14.

Table 14 Component Drug B mAb1 20-100 mg / ml Acetate Polysorbate 20 0.05% Sucrose 8% Arginine pH 5,0

Example 6. The stability of the drug A containing 100 mg / ml mAb1, after production in a bottle and syringes

Preparation A (see table 9), containing 100 mg / ml mAb1, was prepared in a 2 ml glass vial and in two different syringes: standard and low tungsten. The preparations were stored at 5, 25 and 37 ° C for various periods of time. The stability of mAb1 after storage was determined using size exclusion HPLC and cation exchange HPLC. The results are presented in table 15. (An increase in the percentage of the acid variant is consistent with deamidation of the antibody and is therefore considered an undesirable phenomenon with respect to the pharmaceutical preparations of the present invention). As shown in this table, formulation A, containing 100 mg / ml mAb1, stored at 5 ° C in a glass vial or syringe, remained relatively stable for at least 18 months and was slightly less stable at 25 ° C or 37 ° C at later verified times.

Table 15 Stability of drug A containing 100 mg / ml mAb1 in vial and syringe 2 ml glass bottle Standard syringe Low Tungsten Syringe Temperature Time % native form (exclusion HPLC) % acid variant (cation exchange HPLC) % native form (exclusion HPLC) % acid variant (cation exchange HPLC) % native form (exclusion HPLC) % acid variant (cation exchange HPLC) - Start 98.2 14,4 98.2 14.1 98.2 14.3 37 ° C 7
days 97.6 13.9 97.6 14.0 97.8 13.9 37 ° C 14 days 97.2 14.7 97.3 14.6 97.3 14.7 37 ° C 28 days 96.7 15.7 96.6 15,5 96.5 15,5 25 ° C one
month 97.7 13.1 97.5 13.5 97.9 13.3 25 ° C 3
of the month 97.3 14.9 97.2 14.9 97.1 14.8 25 ° C 6
of the month 96.9 17.4 97.1 16.9 96.7 17.4 5 ° C one
month 97.9 14.8 98.1 13.7 98.0 14.0 5 ° C 3
of the month 97.8 13.7 97.6 13.9 97.7 13.5 5 ° C 6
months 98.2 14.0 98.1 13.5 98.0 13.7 5 ° C 9
months 97.4 13,4 97.6 13.1 97.6 13,2 5 ° 12 months 98.1 13.8 98.1 13.5 98.2 13.1 5 ° C 18 months 97.6 14.0 97.6 14.1 97.7 14.0

Example 7. The stability of the drug, containing 100 mg / ml mAb1, after production in a bottle and syringes

Formulation B (see table 14) containing 100 mg / ml mAb1 was prepared in a 2 ml glass vial and in two different syringes: standard and low tungsten. The preparations were stored at 5, 25 and 37 ° C for various periods of time. The stability of mAb1 after storage was determined using size exclusion HPLC and cation exchange HPLC. The results are presented in table 16. (As noted earlier, the increase in the percentage of the acid variant is in accordance with the deamidation of the antibody and is therefore considered an undesirable phenomenon with respect to the pharmaceutical preparations of the present invention). As shown in this table, preparation B containing 100 mg / ml mAb1, stored at 5 ° C in a glass vial or syringe, remained relatively stable for at least 12 months and was slightly less stable at 25 ° C or 37 ° C at all verified times.

Table 16 Stability of drug B containing 100 mg / ml mAb1 in vial and syringe 2 ml glass bottle Standard syringe Low Tungsten Syringe Pace. Time % native form (exclusion HPLC) % acid variant (cation exchange HPLC) % native form (exclusion HPLC) % acid variant (cation exchange HPLC) % native form (exclusion HPLC) % acid variant (cation exchange HPLC) - Start 98.2 14.2 98.1 14,4 98.2 14.3 37 ° C 7
days 97.4 14.3 97.4 14.3 97.6 14.1 37 ° C 14 days 97.0 14.9 96.9 14.8 96.9 14.8 37 ° C 28 days 96.4 16.3 96.4 16,2 96.4 15.9 25 ° C one
month 97.7 13.6 97.8 13.7 97.7 13.6 25 ° C 3
of the month 96.7 14.7 96.9 14.7 96.8 14.8 25 ° C 6
months 96.6 15.4 96.6 15,5 96.6 15.6 5 ° C one
month 98.1 13.6 98.0 13.7 98.1 13.5 5 ° C 2
of the month 97.8 14.3 97.9 14.3 97.9 14.2 5 ° C 3
of the month 97.7 14.3 97.3 14,4 97.4 14.2 5 ° C 6
months 97.4 14,4 97.5 14.5 97.6 14.5 5 ° C 9
months 97.8 14.2 97.8 14.1 97.7 14.1 5 ° C 12 months 98.0 15.1 98.1 14.7 98.2 14.7 5 ° C 18 months 97.7 14.3 97.7 39.8 97.7 14.1

Example 8: Stability of mAb1 Preparations in Prefilled Syringes

A series of experiments were performed to evaluate the stability of various mAb1 preparations in prefilled syringes. For these experiments, various Luer needles and non-removable needles, syringes with the usual tungsten content and low tungsten content were used in combination with various types of cuffs (coated with and without a layer) and screw caps. The stability of the preparations was checked after storage in pre-filled syringes at 37 ° C, 25 ° C and 5 ° C for various periods of time (in the range from 7 days to 6 months, depending on the conditions being studied).

In this example, the stability of the following mAb1 preparations in prefilled syringes was tested: (1) preparation A (see table 9) containing 100 mg / ml mAb1 in a prefilled syringe with a fixed needle # 1 described in table 17; (2) a preparation A containing 100 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 2 described in table 18; (3) a preparation A containing 20 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 1, described in table 19; (4) a preparation A containing 20 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 2 described in table 20; (5) a preparation A containing 100 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 3 described in table 21; and (6) a preparation A containing 6 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 3 described in table 22.

Syringe # 1 is a 1-ml low-tungsten BD syringe with a 29G × ½ inch Physiolis needle; syringe # 2 is a 1 ml Schott syringe with a 29 CF x 1/2 inch SN CF needle; and syringe # 3 is a 1 ml standard Daikyo Seiko CZ® (Crystal Zenith) syringe with a 30G × ½ inch needle.

Stability was evaluated by the following parameters: (a) visual analysis; (b) turbidity (OD _{405 nm} ); (c) percent recovery using reverse phase HPLC; (d) the percentage of the native form of mAb1 using exclusion HPLC; (e) the percentage of the main peak of mAb1 using cation exchange HPLC; and (f) the percentage of the acid variant using cation exchange HPLC.

The results of a representative experiment in which the stability of a preparation A containing 100 mg / ml mAb1 was evaluated in syringe # 1 are presented in table 17 below.

Table 17 The stability of drug A, containing 100 mg / ml mAb1, in a pre-filled syringe with a fixed needle # 1 Description of Syringe # 1: Syringe: 1 ml low-tungsten BD syringe with a 29G × ½-inch needle Physiolis Cuff: Hypak FluroTec® 4023/50 Screw cap: Bd 260 Siliconization: Spraying Pace. Time Visual analysis Turbidity (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 98.2 40,2 14.2 37 ° C 7 days Good luck 0.00 101 97.8 37.6 13.9 37 ° C 14 days Good luck 0.00 one hundred 97.3 37.0 14.7 37 ° C 28 days Good luck 0.00 96 96.5 34.9 15,5 25 ° C 1 month Good luck 0.00 97 97.9 36.9 13.3 25 ° C 2 months Good luck 0.00 103 97.0 38.6 14.9 25 ° C 3 months Good luck 0.00 one hundred 97.1 35.5 14.8 25 ° C 6 months Good luck 0.00 104 96.7 36.8 17.4 5 ° C 1 month Good luck 0.00 96 98.0 39.0 13.8 5 ° C 2 months Good luck 0.00 103 97.8 39.3 13.6 5 ° C 3 months Good luck 0.00 one hundred 97.7 39.2 13.5 5 ° C 6 months Good luck 0.00 one hundred 98.0 39.2 13.7 5 ° C 9 months Good luck 0.00 one hundred 97.6 39.1 13,2 5 ° C 12 months Good luck 0.00 102 98.2 40.9 13.1 5 ° C 18 months Good luck 0.00 one hundred 97.7 39.6 14.0

The results of a representative experiment in which the stability of a preparation A containing 100 mg / ml mAb1 was evaluated in syringe # 2 are shown in table 18 below.

Table 18 The stability of drug A, containing 100 mg / ml mAb1, in a pre-filled syringe with a fixed needle # 2 Description of Syringe # 2: Syringe: 1 ml Schott syringe with 29 CF × 1/2 inch SN CF needle Cuff: West FluroTec® 4023/50 Screw cap: Stelmi 4800 w / RNS Siliconization: Spraying Pace. Time Visual analysis (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 98.3 40.3 14.2 37 ° C 7 days Good luck 0.00 102 97.5 37.8 14.0 37 ° C 14 days Good luck 0.00 102 97.3 37,4 14.7 37 ° C 28 days Good luck 0.00 97 96.6 34.6 15.4 25 ° C 1 month Good luck 0.00 98 97.8 36.8 13,4 25 ° C 2 months Good luck 0.00 103 97.1 38.8 14.9 25 ° C 3 months Good luck 0.00 101 97.1 35.6 14.8 25 ° C 6 months Good luck 0.00 103 96.8 37,4 17.5 5 ° C 1 month Good luck 0.00 97 97.8 39.8 13.8 5 ° C 2 months Good luck 0.00 103 97.8 39,4 13.9 5 ° C 3 months Good luck 0.00 101 97.6 39.8 13.8 5 ° C 6 months Good luck 0.00 101 98.1 40,0 13.9 5 ° C 9 months Good luck 0.00 102 97.6 39.7 13,4 5 ° C 12 months Good luck 0.00 one hundred 98.2 41.1 13.0 5 ° C 18 months Good luck 0.00 102 97.7 39,4 13.9

The results of a representative experiment in which the stability of a preparation A containing 20 mg / ml mAb1 was evaluated in syringe # 1 are shown in table 19 below.

Table 19 The stability of drug A, containing 20 mg / ml mAb1, in a pre-filled syringe with a fixed needle # 1 Description of Syringe # 1: Syringe: 1 ml low-tungsten BD syringe with a 29G × ½-inch needle Physiolis Cuff: Hypak FluroTec® 4023/50 Screw cap: Bd 260 Siliconization: Spraying Pace. Time Visual analysis Turbidity (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 98.9 44.5 15.9 37 ° C 7 days Good luck 0.00 one hundred 98.4 42.1 15.7 37 ° C 14 days Good luck 0.00 99 98.3 41.8 16,2 37 ° C 28 days Good luck 0.00 98 97.6 39.2 17.5 25 ° C 1 month Good luck 0.00 96 98.4 41,4 15.3 25 ° C 2 months Good luck 0.00 one hundred 98.1 41.8 16,0 25 ° C 3 months Good luck 0.00 99 98.0 42.7 17.0 25 ° C 6 months Good luck 0.01 99 98.0 42.0 19,4 5 ° C 1 month Good luck 0.00 97 98.2 44.0 15.7 5 ° C 2 months Good luck 0.00 99 98.5 44.5 15.8 5 ° C 3 months Good luck 0.00 one hundred 98.4 44,4 15.8 5 ° C 6 months Good luck 0.00 99 98.6 44.1 15.6 5 ° C 9 months Good luck 0.00 101 98.2 44.0 15.1 5 ° C 12 months Good luck 0.00 101 98.5 45.4 15.9 5 ° C 18 months Good luck 0.00 101 98.2 43.5 15.3

The results of a representative experiment in which the stability of a preparation A containing 20 mg / ml mAb1 was evaluated in syringe # 2 are presented in table 20 below.

Table 20 The stability of drug A, containing 20 mg / ml mAb1, in a pre-filled syringe with a fixed needle # 2 Description of Syringe # 2: Syringe: 1 ml Schott syringe with 29 CF × 1/2 inch SN CF needle Cuff: West FluroTec® 4023/50 Screw cap: Stelmi 4800 w / RNS Siliconization: Spraying Pace. Time Visual analysis Turbidity (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 98.9 44.5 15.9 37 ° C 7 days Good luck 0.00 101 98.4 42.1 15.6 37 ° C 14 days Good luck 0.00 one hundred 98.2 41.8 16,2 37 ° C 28 days Good luck 0.00 98 97.6 39.3 17.4 25 ° C 1 month Good luck 0.00 97 98.3 41.5 15,2 25 ° C 2 months Good luck 0.00 99 98.2 41.9 16,0 25 ° C 3 months Good luck 0.00 one hundred 98.0 42.7 16.9 25 ° C 6 months Good luck 0.00 99 97.9 42.1 19.5 5 ° C 1 month Good luck 0.00 96 98.4 44.7 15.6 5 ° C 2 months Good luck 0.00 99 98.5 44.3 15.9 5 ° C 3 months Good luck 0.00 99 98.4 44.7 15.7 5 ° C 6 months Good luck 0.00 one hundred 98.7 44,2 15,5 5 ° C 9 months Good luck 0.00 102 98.4 43.8 15.1 5 ° C 12 months Good luck 0.00 102 98.6 45,2 15.9 5 ° C 18 months Good luck 0.00 102 98.3 43,2 15,2

The results of a representative experiment in which the stability of a preparation A containing 100 mg / ml mAb1 was evaluated in syringe # 3 are presented in table 21 below.

Table 21 The stability of drug A containing 100 mg / ml mAb1 in a pre-filled syringe with a fixed needle # 3 Description of Syringe # 3: Syringe: standard 1-ml syringe Daikyo Seiko CZ with a needle size 30G × ½ inch Cuff: Daikyo D-21-6-1 FluroTec® Coated Screw cap: 7028 Siliconization: not applicable to this case Pace. Time Visual analysis Turbidity (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 97.5 38.5 13.6 37 ° C 7 days Good luck 0.00 101 97.5 40,0 14.0 37 ° C 14 days Good luck 0.00 102 97.3 39.5 15,2 37 ° C 28 days Good luck 0.00 101 96.4 38.3 15.7 25 ° C 1 month Good luck 0.00 105 97.8 38.3 13.6 25 ° C 2 months Good luck 0.00 102 97.2 37,4 14.9 25 ° C 3 months Good luck 0.00 102 97.2 37,2 14.6 5 ° C 1 month Good luck 0.00 104 98.2 38.1 13.3 5 ° C 2 months Good luck 0.00 101 97.9 38.1 13,4 5 ° C 3 months Good luck 0.00 101 97.8 38,4 13.1 5 ° C 6 months Good luck 0.00 106 97.8 39.6 13,4 5 ° C 9 months Good luck 0.00 105 97.9 40.8 13,4 5 ° C 12 months Good luck 0.00 112 97.6 38.3 13.5

The results of a representative experiment in which the stability of a preparation A containing 6 mg / ml mAb1 was evaluated in syringe # 3 are presented in table 22 below.

Table 22 The stability of drug A, containing 6 mg / ml mAb1, in a pre-filled syringe with a fixed needle # 3 Description of Syringe # 3: Syringe: standard 1-ml syringe Daikyo Seiko CZ with a needle size 30G × ½ inch Cuff: Daikyo D-21-6-1 FluroTec® Coated Screw cap: 7028 Siliconization: N is not applicable to this case. Pace. Time Visual analysis Turbidity (OD _{405 nm} ) % extraction % native form mAb1 (exclusion HPLC) % of the main peak (cation exchange HPLC) % acid variant (cation exchange HPLC) - Start Good luck 0.00 one hundred 98.8 45.6 16.8 37 ° C 7 days Good luck 0.00 101 98.7 46.2 17,2 37 ° C 14 days Good luck 0.00 101 98.5 45.8 18.2 37 ° C 28 days Good luck 0.00 101 98.3 44.5 19.8 25 ° C 1 month Good luck 0.00 106 98.8 45.7 17.3 25 ° C 2 months Good luck 0.00 103 98.8 44.3 18.1 25 ° C 3 months Good luck 0.00 101 98.3 42.9 18.6 5 ° C 1 month Good luck 0.00 105 99.0 46.0 16.6 5 ° C 2 months Good luck 0.00 102 98.8 45.6 16.8 5 ° C 3 months Good luck 0.00 101 98.8 45.7 16.3 5 ° C 6 months Good luck 0.00 105 98.5 46.7 16.5 5 ° C 9 months Good luck 0.00 106 98.8 47.8 17,2 5 ° C 12 months Good luck 0.00 115 97.8 47.6 17.5

The results of this series of experiments show that various preparations remain relatively stable in pre-filled syringes, especially when stored at temperatures of 25 ° C or lower for one month or longer.

Example 9: Stability of preparations containing low concentrations of mAb1 in glass vials

The stability of mAb1 at concentrations of 0.2, 0.5, 1, and 2 mg / ml is evaluated in glass bottles during real-time studies and during accelerated studies, the results of which are currently presented in Tables 23-27. In the case of these experiments, the stability of mAb1 is tested in Schott type 1 borosilicate glass vials. The mAb1 preparation used in Examples 9 to 12 is similar to that described in Table 9, except that the antibody concentrations used are lower than those previously studied and vary throughout the study periods. The exact concentrations of mAb1 used in each experiment are noted in each table below. The stability of the preparations was checked after storage in glass bottles at 45 ° C, 25 ° C and 5 ° C for various periods of time (in the range from 7 days to 6 months, depending on the conditions being studied).

The results to date indicate increased degradation (precipitation, aggregation, cleavage, and charge-different options) of mAb1 preparations at a concentration of 0.2 and 0.5 mg / ml after incubation at 45 ° C for 7-14 days. In addition, there was an increase in aggregation in mAb1 preparations at all concentrations of ≤2 mg / ml after incubation at 45 ° C for 28 days (> 15% versus ~ 2% in the case of mAb1 preparations at a concentration of ≥6 mg / ml). Precipitation was noted when the drug mAb1 at a concentration of 0.2 mg / ml was stored at 5 ° C for 6 months. Significant degradation / precipitation was not observed when mAb1 preparations at concentrations of 0.5, 1, and 2 mg / ml were stored at 5 ° C for 6 months.

Table 23 Stability of mAb1 at low concentrations in glass vials (incubation at 45 ° C) Time (days) % recovered mAb1 (reverse phase HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 one hundred one hundred one hundred one hundred 7 91 97 103 109 fourteen 67 94 101 108 28 40 82 one hundred 106 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

Table 24 Stability of mAb1 at low concentrations in glass vials (incubation at 25 ° C) Time (days) % of the recovered native form mAb1 (exclusion HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 98.1 98.4 98.6 98.5 7 98.3 98.6 98.6 98.7 fourteen 98.1 98.7 98.6 98.7 28 97.6 98.5 98.6 98.7 56 92.2 96.6 98.1 98.5 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

Table 25 Stability of mAb1 at low concentrations in glass vials (incubation at 5 ° C) Time (months) % recovered mAb1 (reverse phase HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 one hundred one hundred one hundred one hundred one 101 97 105 109 2 99 one hundred 107 110 3 105 99 105 111 6 80 97 102 106 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

Table 26 Stability of mAb1 at low concentrations in glass vials (incubation at 5 ° C) Time (months) % of the recovered native form mAb1 (exclusion HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 98.1 98.4 98.6 98.5 one 97.7 98.6 98.7 98.8 2 97.8 98.3 98.3 98.7 3 98.2 98.7 98.7 98.3 6 97.6 98.4 98.6 98.5 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

Table 27
Stability of mAb1 at low concentrations in PFS syringes or glass vials (incubation at 45 ° C)
A. Glass Bottles Time (days) % of the recovered native form mAb1 (exclusion HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 98.1 98.4 98.6 98.5 7 90,4 97.2 98.3 98.2 fourteen 76.1 92.0 96.1 97.8 28 42.9 51.0 83.7 81.7 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

B. Syringe.

Time (days) % of the recovered native form mAb1 (exclusion HPLC) MAb concentration 1 (mg / ml vial) 0.2 0.5 1,0 2.0 0 98.1 98.6 98.5 98.4 7 97.6 98.1 98.2 98.1 fourteen 97.9 97.9 97.8 97.8 28 96.9 96.8 96.7 96.6 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Example 10: Stability of preparations containing low mAb1 concentrations in BD and Ompi syringes

The stability of mAb1 at concentrations of 0.2, 0.5, 1, and 2 mg / ml was evaluated in BD and Ompi syringes during real-time studies and during accelerated studies. Both syringes are 1-ml syringes made with a low tungsten content and have a 27G × ½ inch thin-walled, non-removable needle. The BD syringe contains 0.8 mg of silicone sprayed on top of the rim. The Ompi syringe contains 0.5 mg of silicone, which is applied using the technology using the lowering nozzles, which provides a more uniform coating along the entire length of the syringe barrel. The stability of the preparations was checked after storage in both pre-filled syringes at 45 ° C, 25 ° C and 5 ° C for various periods of time (in the range from 7 days to 6 months, depending on the conditions being studied).

The results, which are presented in tables 28-30, indicate that the stability of mAb1 at 45 ° C in a BD syringe is significantly higher than in a type 1 borosilicate glass vial, as determined by analysis using reverse phase HPLC (precipitation ) and exclusion HPLC (aggregation and cleavage options).

The stability of mAb1 at 45 ° C in an Ompi syringe was higher than in type 1 borosilicate glass vials, but significantly less than during incubation in a BD syringe. These data suggest that silicone can block the interaction of the drug with a glass surface, thereby increasing stability. Since the BD syringe contains more silicone throughout the syringe’s glass cylinder than the Ompi syringe, this may explain why the drug is more stable in the BD syringe than in the Ompi syringe.

No significant degradation was observed when mAb1 was stored in BD and Ompi syringes at 5 ° C or 25 ° C for 6 months, as determined by analysis using exclusion HPLC and cation exchange HPLC.

Table 28
Stability of mAb1 at a concentration of 0.6 mg / ml in BD syringes, Ompi syringe or glass vials (incubation at 45 ° C) Time (days) % of the recovered native form mAb1 (exclusion HPLC) Type 1 glass bottle Syringe BD Syringe Ompi 0 98.6 98.6 98.9 7 98.2 98.4 98.5 fourteen 97.7 98.0 98.5 28 66.1 97.1 84.5 54 - 95.6 24.5 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 29 Stability of mAb1 at low concentrations in BD and Ompi syringes (incubation at 5 ° C, exclusion HPLC) Time (months) % of the recovered native form mAb1 (exclusion HPLC) The concentration of mAb1 (mg / ml) Bottle Bd Ompi 0.2 0.6 0.2 0.6 0.2 0.6 0 98.6 98.6 98.4 98.6 98.7 98.9 one 98.7 99.0 98.5 98.9 98.5 98.9 2 98.3 98.9 98.7 98.9 98.6 98.9 3 98.4 98.6 98.4 98.7 98.3 98.7 6 98.3 98.6 98.3 98.7 98.2 98.7 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 30 Stability of mAb1 at low concentrations in vial, syringe BD and CZ (incubation at 45 ° C, reverse phase HPLC) % recovered mAb1 (reverse phase HPLC) The concentration of mAb1 (mg / ml) Time (days) Bottle Bd Cz 0.2 0.6 0.2 0.6 0.2 0.6 0 one hundred one hundred one hundred one hundred one hundred one hundred 7 92 103 96 107 99 106 fourteen 88 96 96 99 104 one hundred 28 39 94 85 99 104 101 42 - - 61 - 94 - 56 - - 55 - 96 - 62 - - - 88 - 98 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Example 11: Stability of a preparation containing low concentrations of mAb1 in alternative storage devices.

Studies on the stability of mAb1 in other storage devices, including West CZ syringes (made from cyclic polyolefin), plus® Schott type 1 glass bottles (with a 100-200 nm layer of SiO ₂ on the inner surface of type 1 borosilicate glass bottles) and glass bottles Schott type 1 borosilicate glass with nitrogen-filled free space (air removed from the free space of the vial and replaced with N ₂ gas).

The results presented in tables 31-36 indicate that the stability of mAb preparations at concentrations of 0.2 and 0.6 mg / ml at 45 ° C in CZ syringes was significantly higher than in BD syringes as determined by HPLC reverse phase and size exclusion HPLC.

In addition, the stability of mAb1 preparations at concentrations of 0.2 and 0.6 mg / ml at 45 ° C in Schott plus® type 1 glass vials was significantly higher than in type 1 vials, as determined by reverse phase HPLC and exclusion HPLC.

Significant degradation, determined using exclusion HPLC, was observed in the case of mAb1 at a concentration of 0.2 mg / ml in type 1 glass vials after incubation at 25 ° C or 5 ° C for 3 months. Significant degradation was not observed in the case of mAb1 at a concentration of 0.2 mg / ml in plus Schott type 1 glass vials after incubation at 25 ° C or 5 ° C for 3 months.

Stability at 45 ° C was significantly higher in the case of mAb1 preparations at a concentration of 0.2 mg / ml in bottles of glass type 1 with an upper nitrogen layer than in bottles of glass type 1 with air free space, as determined using exclusion HPLC.

The removal of oxygen from the free space had the highest stabilizing effect on mAb1 at a concentration of 0.2 mg / ml, prepared using 0.05% polysorbate 20 (as in preparation A), although an increased rate of degradation was nevertheless observed in comparison with the preparation of mAb1 at a concentration of 0.2 mg / ml without polysorbate 20.

Table 31 Stability of mAb1 at low concentrations in vial, BD and CZ syringes (incubation at 45 ° C, exclusion HPLC) % native form mAb1 (exclusion HPLC) The concentration of mAb1 (mg / ml) Time (days) Bottle Bd Cz 0.2 0.6 0.2 0.6 0.2 0.6 0 98.8 98.7 98.5 98.5 98.7 98.6 7 96.5 98.3 97.6 98.3 97.0 98.3 fourteen 82.0 97.2 95.8 97.2 97.1 97.0 28 58.2 64.5 70.6 95.8 96.1 96.2 42 - - 48.8 - 93.3 - 56 - - 36,4 - 89.6 - 62 - - - 93,4 - 90.3 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 32 Stability of mAb1 at low concentrations in plus® type 1 vial (incubation at 45 ° C, reverse phase HPLC) % recovered mAb1 (reverse phase HPLC) Time (days) The concentration of mAb1 (mg / ml) Type 1 Bottle Plus® bottle type 1 0.2 0.6 0.2 0.6 0 one hundred one hundred one hundred one hundred 7 92 103 99 106 fourteen 88 96 one hundred 99 28 39 94 101 one hundred Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 33 Stability of mAb1 at low concentrations in plus® type 1 vial (incubation at 45 ° C, exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (days) The concentration of mAb1 (mg / ml) Type 1 Bottle Plus® bottle type 1 0.2 0.6 0.2 0.6 0 98.8 98.7 98.7 98.6 7 96.5 98.3 97.3 98.3 fourteen 82.0 97.2 97.4 97.2 28 58.2 64.5 70.6 95.2 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 34 Stability of mAb1 at low concentrations in plus® type 1 vial (incubation at 25 ° C, exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (months) The concentration of mAb1 (mg / ml) Type 1 Bottle Plus® bottle type 1 0.2 0.6 0.2 0.6 0 98.8 98.7 98.7 98.6 one 98.7 98.7 98.6 98.8 2 98.2 98.6 98.2 98.6 3 92.5 97.2 97.5 97.4 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 35 Stability of mAb1 at low concentrations in plus® type 1 vial (incubation at 5 ° C, exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (months) The concentration of mAb1 (mg / ml) Type 1 Bottle Plus® bottle type 1 0.2 0.6 0.2 0.6 0 98.8 98.7 98.7 98.7 one 98.9 98.9 98.5 98.7 2 98.7 98.9 98.6 98.6 3 96.5 97.7 97.9 97.7 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Table 36 Stability of mAb1 at a concentration of 0.2 mg / ml in a type 1 vial with an upper nitrogen layer (incubation at 45 ° C, exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (months) Airspace Nitrogen free space 0 97.5 97.1 7 95.2 95.7 fourteen 84.6 96.6 21 60.9 96.7 28 53.9 96.7 56 - 92.4 Drug: 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0

Example 12: Effect of tungsten on the stability of mAb1 in low concentration

Studies are carried out on the stability of mAb1 after introducing an un-filtered tungsten rod extract. Tungsten rods are used in the manufacturing process of pre-filled syringes to form holes in the Luer syringe. The tungsten rod extract used on the BD production line was prepared using placebo mAb1 (same as preparation A but without mAb1). The non-filtered rod extract contains all varieties of tungsten that may remain in the syringe as impurities (soluble tungsten salts and insoluble tungsten oxides).

The results of exclusion HPLC presented in tables 37 and 38 indicate that the stability at 25 ° C of preparations of mAb1 at a concentration of 0.6 mg / ml containing tungsten levels of ≥500 ppm was less than in a control vial without tungsten . Low tungsten syringes typically contain <500 ppm tungsten, while standard syringes can contain up to 2500 ppm tungsten.

No degradation was observed in the case of Mab1 preparations with a concentration of 0.6 mg / ml with tungsten up to 2500 ppm after incubation at 5 ° C for 6 months.

Table 37 Stability of mAb1 at a concentration of 0.6 mg / ml after storage at 25 ° C in non-filtered extracts of tungsten rods (exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (weeks) Control bottle 50 ppm 100 ppm 500 ppm 1000 ppm 2500 ppm 0 98.7 98.8 98.9 98.8 98.8 98.7 one 98.7 98.6 98.7 98.6 98.7 98.7 2 98.6 98.6 98.6 98.4 98.4 98.4 four 98.5 98.5 98.5 98.6 98.2 98.5 8 98.6 98.7 98.6 98.2 98.3 98.6 26 97.4 97.8 97.5 78.6 76.5 74.8 Preparation: 0.6 mg / ml mAb in 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

Table 38 Stability of mAb1 at a concentration of 0.6 mg / ml after storage at 5 ° C in non-filtered extracts of tungsten rods (exclusion HPLC) % native form mAb1 (exclusion HPLC) Time (weeks) Control bottle 50 ppm 100 ppm 500 ppm 1000 ppm 2500 ppm 0 98.7 98.8 98.9 98.8 98.8 98.7 one 98.5 98.5 98.6 98.7 98.6 98.4 3 97.7 98.0 98.2 98.2 98.2 98.2 6 98.8 98.6 98.7 98.7 98.8 98.4 Preparation: 0.6 mg / ml mAb in 8% sucrose; 0.05% polysorbate 20, 10 mm acetate, pH 5.0.

The specific embodiments described herein do not limit the scope of the present invention. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art based on the above description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims (17)

1. The method of maintaining the stability of the antibodies in the drug, including: storing the antibody preparation for six months or more at 5 ° C or more; where the antibody preparation includes: (i) about 0.1-100 mg / ml of a human antibody that specifically binds to human nerve growth factor (hNGF), where the antibody contains a pair of amino acid sequences of the variable region of the heavy chain and the variable region of the light chain (HCVR / LCVR) selected from a group consisting of: (i) SEQ ID NO: 20/22; and (ii) SEQ ID NO: 4/12; (ii) approximately 0.01-1.0% polysorbate 20; (iii) approximately 1-20% sucrose and (iv) from about 1 mM to about 50 mM acetate; as a result, the preparation in storage maintains such stability that 90% or more of the antibodies are detected by high-resolution liquid chromatography (size exclusion HPLC). 2. The method for maintaining the stability of an antibody according to claim 1, wherein the preparation comprises: (i) approximately 0.2-75 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.02-0.5% polysorbate 20; and (iii) about 5-10% sucrose. 3. The method for maintaining the stability of an antibody according to claim 2, wherein the preparation comprises: (i) approximately 0.6-60 mg / ml of a human antibody that specifically binds to hNGF; (ii) about 0.5% polysorbate 20; and (iii) about 8% sucrose. 4. The method for maintaining the stability of the antibody according to claim 1, wherein the preparation is in a container selected from the group consisting of a glass vial, a glass syringe, a plastic vial, and a plastic syringe. 5. The method of maintaining the stability of the antibody according to claim 4, in which the container is a glass bottle coated with silicon dioxide. 6. The method of maintaining the stability of the antibody according to claim 5, in which the glass vial is filled with the drug, and the remaining free space is filled with an inert gas. 7. The method of maintaining the stability of the antibody according to claim 6, in which the inert gas is selected from the group consisting of argon and nitrogen. 8. The method for maintaining the stability of the antibody according to claim 1, wherein the preparation is in a container selected from the group consisting of an auto-injector and a micro-infusion device. 9. The method of maintaining the stability of the antibody according to claim 4, in which the container is a syringe containing a piston cuff coated with a layer of fluorocarbon. 10. The method of maintaining the stability of the antibody according to claim 1, in which the drug is presented in a syringe containing a low content of tungsten. 11. The method of maintaining the stability of the antibody according to claim 1, in which the drug is presented in a syringe containing a piston cuff coated with a layer of fluorocarbon.